Since we first discovered in 1987 the depletion of mitochondrial GSH (mGSH) status by chronic ethanol intake, considerable progress has been made regarding the molecular mechanism(s) of the defect and its functional impact in alcohol-induced liver damage (ALD). In addition to the altered regulation of mGSH by alcohol, S-adenosyl-L-methionine (SAM) depletion has been also reported and is thought to contribute to the progression of the disease. GSH and SAM may exhibit parallel features as both are found in mitochondria due to specific transport mechanisms. Thus, the present proposal will look at the specific regulation of mitochondrial SAM (mSAM) pool by alcohol and the temporal and functional relationship between mGSH and mSAM by alcohol. 1. - SAM levels and transport in rat liver mitochondria from alcohol-fed rats. Most studies have reported the status of total SAM levels in hepatocellular extracts in patients and experimental models of ALD. We wilt examine the status of mSAM levels and its mitoehondrial transport in chronic alcohol-fed rats. 2. Temporal and functional relationship between mitochondrial GSH and SAM depletion by alcohol: Role of Kupffer cells and liver steatosis. We will examine if mGSH depletion by alcohol precedes or follows that of SAM (total and mSAM) in mitochondria from alcohol fed rats and its relationship with pathology. The role of Kupffer cell activation and liver steatosis on alcohol-mediated mGSH and total SAM (cytosol and mSAM) depletion will be evaluated in alcohol-fed rats and in a model of non-alcohol: hepatic steatosis. 3. Mechanism whereby SAM feeding normalizes the alcohol-altered mitoehondrial membrane fluidity and mGSH transport. Increasing evidence indicate a critical role of cholesterol/phospholipid molar ratio in the regulation of mitochondrial membrane mieroviseosity and mGSH transport, in addition to these changes, we wilt examine the regulation and trafficking of cholesterol into mitoehondria, as well as the fatty acid composition of individual lipids of mitochondrial lipid classes from chronic alcohol-fed rats with or without SAM supplementation. 4. Regulation of MAT1A by glycosphingolipids and sphingomyelinases. TNF and short-chain ceramide have been reported to downregulate the expression of MAT1A. Since sphingomyelinases (SMases) are known to mediate some of the effects of TNF, we will examine the role of individual neutral or acidic SMase, in the regulation of MATIA in primary cultured rat hepatocytes and HepG2 cells exposed to exogenous neutral (NSMase) or acid (ASMase) SMases. In addition, we will assess the role of TNF on MATIA expression in ASMase knockout mice.